Antibiotic macrocycle compounds and methods of manufacture and use thereof

ABSTRACT

The invention encompasses an active metabolite of the 18-membered macrocyclic antimicrobial agents, specifically, a metabolite of tiacumicin B and in certain embodiments, R-Tiacumicin B or and its related compounds. In particular, the invention encompasses a compound that acts as a potent antibiotic agent for the treatment of bacterial infections, specifically GI infections caused by toxin producing strains of  Clostridium difficile  ( C. difficile ) and  Clostridium perfringens  ( C. perfringens ).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/280,148, filed on Oct. 24, 2011, now U.S. Pat. No. 8,445,654which is a continuation application of U.S. application Ser. No.12/324,907 filed Nov. 28, 2008, now U.S. Pat. No. 8,044,030, whichclaims the benefit of U.S. provisional patent application No.60/996,609, filed Nov. 27, 2007, all of which are herein incorporatedherein by reference in their entireties.

This application claims the benefit of U.S. provisional patentapplication No. 60/996,609, filed Nov. 27, 2007, which is incorporatedherein by reference in it entirety.

FIELD OF THE INVENTION

The invention encompasses an active metabolite of the 18-memberedmacrocyclic antimicrobial agents, specifically, a metabolite oftiacumicin B and in certain embodiments, R-Tiacumicin B or and itsrelated compounds. In particular, the invention encompasses a compoundthat acts as a potent antibiotic agent for the treatment of bacterialinfections, specifically GI infections caused by toxin producing strainsof Clostridium difficile (C. difficile) and Clostridium perfringens (C.perfringens).

BACKGROUND OF THE INVENTION

Macrocycles are an important therapeutic class of antibiotics. Thesecompounds are frequently produced as a family of closely relatedbiogenetic congeners. The Tiacumicins are a series of 18-memberedmacrocyclic antibiotics in which the macrocyclic ring is glycosidicallyattached to one or two sugars. As shown in Table 1, a seven-carbon sugar(R¹) is esterified at various positions with small fatty acids. Theother sugar (R²), when present, is esterified with an isomer ofsubstituted benzoic acid such as everninic acid (Journal of LiquidChromatography, 1988, 11: 191-201).

Tiacumicins are a family of related compounds that contain the18-membered ring shown in Formula I below.

At present, several distinct Tiacumicins have been identified and six ofthese (Tiacumicin A-F) are defined by their particular pattern ofsubstituents R¹, R², and R³ (See, e.g., U.S. Pat. No. 4,918,174; J.Antibiotics, 1987, 40: 575-588), as shown in Table 1.

TABLE 1 Substituents Present In Tiacumcins A-F R¹ R² R³ A

H H B

OH C

OH D

OH E

OH F

OH

More recently, a group of related substances were discovered anddescribed in the U.S. patent application publication numberUS2007-105791 and include the following.

Tiacumicins can be produced by bacteria, including Dactylosporangiumaurantiacum subspecies hamdenensis, which may be obtained from the ARSPatent Collection of the Northern Regional Research Center, UnitedStates Department of Agriculture, 1815 North University Street, Peoria,Ill. 61604, accession number NRRL 18085. The characteristics of strainAB 718C-41 are given in J. Antibiotics, 1987, 40: 567-574 and U.S. Pat.No. 4,918,174.

Tiacumicins show activity against a variety of bacterial pathogens andin particular against C. difficile, a Gram-positive bacterium(Antimicrob. Agents Chemother. 1991, 1108-1111). C. difficile is ananaerobic spore-forming bacterium that causes an infection of the bowel.Diarrhea is the most common symptom but abdominal pain and fever mayalso occur. C. difficile is a major causative agent of colitis (i.e.,inflammation of the colon) and diarrhea that may occur followingantibiotic intake. This bacterium is primarily acquired in hospitals andchronic care facilities. Because Tiacumicin B shows promising activityagainst C. difficile, it is expected to be useful in the treatment ofbacterial infections, especially those of the gastrointestinal tract, inmammals. Examples of such treatments include but are not limited totreatment of colitis and treatment of irritable bowel syndrome.Tiacumicins may also find use for the treatment of gastrointestinalcancers.

Tiacumicin antibiotics are described in U.S. Pat. No. 4,918,174 (issuedApr. 17, 1990), J. Antibiotics, 1987, 40: 575-588, J. Antibiotics, 1987,40: 567-574, J. Liquid Chromatography, 1988, 11: 191-201, AntimicrobialAgents and Chemotherapy 1991, 35: 1108-1111, U.S. Pat. No. 5,583,115(issued Dec. 10, 1996), and U.S. Pat. No. 5,767,096 (issued Jun. 16,1998), which are all incorporated herein by reference. Related compoundsare the Lipiarmycin antibiotics (c.f., J. Chem. Soc. Perkin Trans. I,1987, 1353-1359 and J. Antibiotics 1988, 41: 308-315) and theClostomicin antibiotics (J. Antibiotics 1986, 39: 1407-1412), which areall incorporated herein by reference.

SUMMARY OF THIS INVENTION

The invention encompasses a compound of Formula II:

in free form or a pharmaceutically acceptable salt thereof.

In other embodiments, the invention encompasses compositions including acompound of Formula II in free form or a pharmaceutically acceptablesalt thereof. In certain embodiments, the invention encompassescompositions including a therapeutically effective amount of a compoundof Formula II in free form or a pharmaceutically acceptable saltthereof. In certain other embodiments, the compositions further includea pharmaceutically acceptable carrier or excipient.

In another embodiment, the invention encompasses methods of treatingdiseases and/or disorders, for example, infections, for example,bacterial infections caused by gram-positive anaerobes, by administeringto a subject in need thereof a therapeutically effective amount of acomposition including a compound of Formula II in free form or apharmaceutically acceptable salt thereof.

In still other embodiments, the invention encompasses methods oftreating diseases and/or disorders, for example, infections, forexample, bacterial infections caused by gram-positive anaerobes, bycontacting a subject with an effective amount of a compound of FormulaII in free form or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the Comparison of OPT-80 and Formula II fecal levels withMIC values in the presence and absence of 5% fecal matter vs. C.difficile ATCC 700058.

DETAILED DESCRIPTION OF THE INVENTION General Description

The invention generally encompasses pharmaceutical compositionsincluding a compound of formula II:

in free form or in the form of its pharmaceutically acceptable salts.

In certain embodiments, the compound of Formula II is stereomericallypure.

In certain embodiments, the pharmaceutical composition is in the form ofan oral dosage form.

In certain embodiments, the compound of Formula II is present in anamount of about 0.001 mg to about 4000 mg.

In certain embodiments, the oral dosage form is a tablet, capsule, gelcap, solution, syrup, or elixir.

In certain embodiments, the compound of Formula II is present in anamount of about 0.001 mg to about 2000 mg.

In certain embodiments, the compound of Formula II is present in anamount of about 0.01 mg to about 1000 mg.

In certain embodiments, the compound of Formula II is present in anamount of about 0.1 mg to about 800 mg.

In certain embodiments, the compound of Formula II is present in anamount of about 1 mg to about 500 mg.

In certain embodiments, the compound of Formula II is in the form of ahydrochloride salt.

In certain embodiments, the pharmaceutical composition further comprisesa pharmaceutically acceptable carrier or vehicle.

The invention also encompasses methods for treating or preventingbacterial infections in a mammal, which comprises administering to saidmammal a composition comprising a therapeutically or prophylacticallyeffective amount of a compound of Formula II:

in free form or in the form of its pharmaceutically acceptable salts.

In certain embodiments, the therapeutically or prophylacticallyeffective amount is from about 0.001 mg/kg to about 2000 mg/kg.

In certain embodiments, the mammal is human.

In certain embodiments, the route of administration is oral orparenteral.

In certain embodiments, the route of administration is intravenous,intradermal, intramuscular, intraperitoneal, subcutaneous, epidural,intracerebral, or intravaginal.

In certain embodiments, the dosage is 0.001 milligram to 200 milligramsper kilogram of the subject's body weight.

In certain embodiments, the dosage is 0.01 milligrams to 100 milligramsper kilogram of the subject's body weight.

In certain embodiments, the dosage is 0.1 milligrams to 50 milligramsper kilogram of the subject's body weight.

The invention also encompasses methods for treating or preventingbacterial infections in a mammal, which comprises contacting thegastrointestinal tract of said mammal with a compound of Formula II:

in free form or in the form of its pharmaceutically acceptable salts.

DEFINITIONS

As used herein, and unless otherwise indicated, the term“antibiotic-associated condition” refers to a condition resulting whenantibiotic therapy disturbs the balance of the microbial flora of thegut, allowing pathogenic organisms such as enterotoxin producing strainsof C. difficile, S. aureus and C. perfringens to flourish. Theseorganisms can cause diarrhea, pseudomembranous colitis, and colitis andare manifested by diarrhea, urgency, abdominal cramps, tenesmus, andfever among other symptoms. Diarrhea, when severe, causes dehydrationand the medical complications associated with dehydration.

As used herein, and unless otherwise indicated, the term “asymmetricallysubstituted” refers to a molecular structure in which an atom havingfour tetrahedral valences is attached to four different atoms or groups.The commonest cases involve the carbon atom. In such cases, two opticalisomers (D- and L-enantiomers or R- and S-enantiomers) per carbon atomresult which are nonsuperposable mirror images of each other. Manycompounds have more than one asymmetric carbon. This results in thepossibility of many optical isomers, the number being determined by theformula 2^(n), where n is the number of asymmetric carbons.

As used herein, and unless otherwise indicated, the terms“biohydrolyzable carbamate,” “biohydrolyzable carbonate,”“biohydrolyzable ureide” and “biohydrolyzable phosphate” mean acarbamate, carbonate, ureide and phosphate, respectively, of a compoundthat either: 1) does not interfere with the biological activity of thecompound but can confer upon that compound advantageous properties invivo, such as uptake, duration of action, or onset of action; or 2) isbiologically inactive but is converted in vivo to the biologicallyactive compound. Examples of biohydrolyzable carbamates include, but arenot limited to, lower alkylamines, substituted ethylenediamines,aminoacids, hydroxyalkylamines, heterocyclic and heteroaromatic amines,and polyether amines.

As used herein, and unless otherwise indicated, the term“biohydrolyzable ester” means an ester of a compound that either: 1)does not interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters,and choline esters.

As used herein, and unless otherwise indicated, the term“biohydrolyzable amide” means an amide of a compound that either: 1)does not interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable amides include, but are not limited to,lower alkyl amides, .alpha.-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides.

As used herein, and unless otherwise indicated, the term “broth” as usedherein refers to the fluid culture medium as obtained during or afterfermentation. Broth comprises a mixture of water, the desiredantibiotic(s), unused nutrients, living or dead organisms, metabolicproducts, and the adsorbent with or without adsorbed product.

As used herein, and unless otherwise indicated, the term “compound” or“compounds of the invention” are used interchangeably and refer to acompound of Formula II, Formula IIa, salts, stereoisomers, mixtures ofstereoisomers, and prodrugs thereof.

As used herein, and unless otherwise indicated, the term “excipient”refers to an inert substance added to a pharmacological composition tofurther facilitate administration of a compound. Examples of excipientsinclude but are not limited to, calcium carbonate, calcium phosphate,various sugars and types of starch, cellulose derivatives, gelatin,vegetable oils and polyethylene glycols.

As used herein, and unless otherwise indicated, the term “halogen”includes F, Cl, Br and I.

As used herein, and unless otherwise indicated, the term “hydrate” meansa compound of the present invention or a salt thereof that furtherincludes a stoichiometric or non-stoichiometric amount of water bound bynon-covalent intermolecular forces.

As used herein, and unless otherwise indicated, the term “isomericmixture” means a mixture of two or more configurationally distinctchemical species having the same chemical formula. An isomeric mixtureis a genus comprising individual isomeric species. Examples of isomericmixtures include stereoisomers (enantiomers and diastereomers),regioisomers, as might result for example from a pericyclic reaction.The compounds of the present invention comprise asymmetricallysubstituted carbon atoms. Such asymmetrically substituted carbon atomscan result in mixtures of stereoisomers at a particular asymmetricallysubstituted carbon atom or a single stereoisomer. As a result, racemicmixtures, mixtures of diastereomers, as well as single diastereomers ofthe compounds of the invention are included in the present invention.

As used herein, and unless otherwise indicated, the term “lower alkyl,”alone or in combination, refers to an optionally substitutedstraight-chain or optionally substituted branched-chain having from 1 toabout 8 carbons (e.g., C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈,), more preferably1 to 4 carbons (e.g., C₁, C₂, C₃, C₄,). Examples of alkyl radicalsinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl. A “lower alkyl” is generally a shorter alkyl,e.g., one containing from 1 to about 4 carbon atoms (e.g., C₁, C₂, C₃,C₄,).

As used herein, and unless otherwise indicated, the term “macrocycles”refers to organic molecules with large ring structures usuallycontaining over 10 atoms.

As used herein, and unless otherwise indicated, the term “18-memberedmacrocycles” refers to organic molecules with ring structures containing18 atoms.

As used herein, and unless otherwise indicated, the term “membered ring”can embrace any cyclic structure, including carbocycles and heterocyclesas described above. The term “membered” is meant to denote the number ofskeletal atoms that constitute the ring. Thus, for example, pyridine,pyran and thiopyran are 6 membered rings and pyrrole, furan, andthiophene are 5 membered rings.

As used herein, and unless otherwise indicated, the term “MIC” or“minimum inhibitory concentration” refers to the lowest concentration ofan antibiotic that is needed to inhibit growth of a bacterial isolate invitro. A common method for determining the MIC of an antibiotic is toprepare several tubes containing serial dilutions of the antibiotic,that are then inoculated with the bacterial isolate of interest. The MICof an antibiotic can be determined from the tube with the lowestconcentration that shows no turbidity (no growth).

As used herein, and unless otherwise indicated, the term “MIC₅₀” refersto the lowest concentration of antibiotic required to inhibit the growthof 50% of the bacterial strains tested within a given bacterial species.

As used herein, and unless otherwise indicated, the term “MIC₉₀” refersto the lowest concentration of antibiotic required to inhibit the growthof 90% of the bacterial strains tested within a given bacterial species.

As used herein, and unless otherwise indicated, the term “OPT-80” refersto a preparation containing R-Tiacumicin B and Tiacumicin B relatedcompounds (including, but not limited to, Tiacumicins, Lipiarmycin A4and C-19 Ketone). Preparations of this type are described in detail inPCT application PCT/US03/21977, having an international publicationnumber of WO 2004/014295 A2 and which preparations and are incorporatedhere by reference.

As used herein, and unless otherwise indicated, the term“pharmaceutically acceptable carrier” refers to a carrier or diluentthat is pharmaceutically acceptable.

As used herein, and unless otherwise indicated, the term“pharmaceutically acceptable salts” refers to those derived frompharmaceutically acceptable inorganic and organic bases. Salts derivedfrom appropriate bases include alkali metal (e.g., sodium or potassium),alkaline earth metal (e.g., magnesium), ammonium and N(C₁-C₄ alkyl)₄ ⁺salts, and the like. Illustrative examples of some of these includesodium hydroxide, potassium hydroxide, choline hydroxide, sodiumcarbonate, and the like. The term “pharmaceutically acceptable salt”also refers to salts prepared from pharmaceutically acceptable non-toxicacids, including inorganic acids and organic acids. Suitable non-toxicacids include inorganic and organic acids such as, but not limited to,acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic,citric, ethenesulfonic, formic, fumaric, furoic, gluconic, glutamic,glucorenic, galacturonic, glycidic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phenylacetic, propionic, phosphoric,salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid,p-toluenesulfonic and the like. Particularly preferred are hydrochloric,hydrobromic, phosphoric, and sulfuric acids, and most particularlypreferred is the hydrochloride salt.

As used herein, and unless otherwise indicated, the term “pharmaceuticalcomposition” refers to a composition of the R-Tiacumicin describedherein, or physiologically acceptable salts thereof, with other chemicalcomponents, such as physiologically acceptable carriers and/orexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to a mammal, including humans.

As used herein, and unless otherwise indicated, the term“physiologically acceptable carrier” is used synonymously with the term“pharmaceutically acceptable carrier” and refers to a carrier or diluentthat does not cause significant irritation to an organism and does notabrogate the biological activity and properties of the administeredcompound.

As used herein, and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide thecompound. Examples of prodrugs include, but are not limited to,compounds that comprise biohydrolyzable moieties such as biohydrolyzableamides, biohydrolyzable esters, biohydrolyzable carbamates,biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzablephosphate analogues. Other examples of prodrugs include compounds thatcomprise —NO, —NO₂, —ONO, or —ONO₂ moieties. When used to describe acompound of the invention, the term “prodrug” may also to be interpretedto exclude other compounds of the invention for example racemates.

As used herein, and unless otherwise indicated, the term“pseudomembranous colitis” or “enteritis” refers to the formation ofpseudomembranous material (i.e., material composed of fibrin, mucous,necrotic epithelial cells and leukocytes) due to inflammation of themucous membrane of both the small and large intestine.

As used herein, and unless otherwise indicated, the term “R” and “S”configuration, as used herein, are as defined by the IUPAC 1974Recommendations for Section E, Fundamental Stereochemistry, Pure Appl.Chem. (1976) 45, 13-30. Chiral molecules can be named based on theatomic numbers of the atoms or groups of atoms, the ligands that areattached to the chiral center. The ligands are given a priority (thehigher the atomic number the higher the priority) and if the prioritiesincrease in a clockwise direction, they are said to be R-configuration.Otherwise, if they are prioritized in a counterclockwise direction theyare said to be S-configuration.

As used herein, and unless otherwise indicated, the term “R-TiacumicinB” refers to the optically pure (R)-isomer of Tiacumicin B with an(R)-hydroxy group at C-19, as shown below:

As used herein, and unless otherwise indicated, the term “stereoisomers”refers to compounds whose molecules have the same number and kind ofatoms and the same atomic arrangement, but differ in their spatialarrangement.

As used herein, and unless otherwise indicated, the terms “opticallypure,” “stereomerically pure,” and “substantially stereomerically pure”are used interchangeably and mean one stereoisomer of a compound or acomposition that comprises one stereoisomer of a compound and issubstantially free of other stereoisomer(s) of that compound. Forexample, a stereomerically pure compound or composition of a compoundhaving one chiral center will be substantially free of the oppositeenantiomer of the compound. A stereomerically pure compound orcomposition of a compound having two chiral centers will besubstantially free of other diastereomers of the compound. A typicalstereomerically pure compound comprises greater than about 80% by weightof one stereoisomer of the compound and less than about 20% by weight ofother stereoisomers of the compound, more preferably greater than about90% by weight of one stereoisomer of the compound and less than about10% by weight of the other stereoisomers of the compound, even morepreferably greater than about 95% by weight of one stereoisomer of thecompound and less than about 5% by weight of the other stereoisomers ofthe compound, and most preferably greater than about 97% by weight ofone stereoisomer of the compound and less than about 3% by weight of theother stereoisomers of the compound.

As used herein, and unless otherwise indicated, the term “subject”refers to a human or animal in need of medical treatment. For thepurposes of this invention, human subjects are typicallyinstitutionalized in a primary medical care facility such as a hospitalor nursing home. However, treatment of a disease associated with the useof antibiotics or cancer chemotherapies or antiviral therapies can occuron an outpatient basis, upon discharge from a primary care facility, orcan be prescribed by a physician for home-care, not in association witha primary medical care facility. Animals in need of medical treatmentare typically in the care of a veterinarian.

As used herein, and unless otherwise indicated, the term “sugar”generally refers to mono-, di- or oligosaccharides. A saccharide may besubstituted, for example, glucosamine, galactosamine, acetylglucose,acetylgalactose, N-acetylglucosamine, N-acetyl-galactosamine,galactosyl-N-acetylglucosamine, N-acetylneuraminic acid (sialic acid),etc., as well as sulfated and phosphorylated sugars. For the purposes ofthis definition, the saccharides are in their pyranose or furanose form.

As used herein, and unless otherwise indicated, the term “Tiacumicin” asused herein refers to a family of compounds all of which comprise the18-membered macrocycle shown below in Formula I:

As used herein, and unless otherwise indicated, the term “yield” as usedherein refers to an amount of crude Tiacumicin re-constituted inmethanol to the same volume as the original fermentation broth. Yield isdetermined using standard HPLC techniques. Yield is reported in units ofmg/L.

Compound of the Invention

The compound of the invention is a metabolite of Tiacumicin B having thestructure of Formula II, wherein the isopropylcarboxy group on the sugarof Tiacumicin B has been cleaved:

This invention relates to a pharmaceutical compound of Formula II infree form or in the form of its pharmaceutically acceptable salts.

In certain embodiments, the compound of the invention is a metabolite ofR-Tiacumicin B, (i.e., the compound of Formula II).

In certain embodiments, the compound of Formula II is racemic and can berepresented by the compound of Formula IIa.

In other embodiments, the compound of Formula II is substantiallystereomerically pure and can be illustrated by the compound of FormulaII.

The invention also encompasses pharmaceutically acceptable salts of themetabolite of tiacumicin B.

Therapeutic and Prophylactic Uses of the Compound of the Invention

The invention encompasses a compound of Formula II that is effective intreating disorders associated with bacterial infections, specifically GIinfections caused by toxin producing strains of Clostridium difficile(C. difficile) and Clostridium perfringens (C. perfringens).

In another embodiment of the invention is the selective effect of thecompound of Formula II on C. difficile similar to that of the parentcompound, OPT-80, and was as active as vancomycin against C. difficile.

In other embodiments, the invention encompasses methods of treatingantibiotic-associated conditions including administering atherapeutically effective amount of a compound of Formula II or FormulaIIa or a salt thereof or a composition comprising such compound to asubject in need thereof.

In other embodiments, the invention encompasses methods of treating acondition resulting when antibiotic therapy disturbs the balance of themicrobial flora of the gut, allowing pathogenic organisms such asenterotoxin producing strains of C. difficile, S. aureus and C.perfringens to flourish. The method includes administering atherapeutically effective amount of a compound of Formula II or FormulaIIa or a salt thereof or a composition comprising such compound to asubject in need thereof.

In other embodiments, the invention encompasses methods of treatingdisorders associated with the abnormal growth of C. difficile, S. aureusand C. perfringens in the intestinal tract and in certain embodiments ina subject taking antibiotics or other antimicrobial drugs. Disordersthat can be treated or prevented by the present methods include symptomsranging from diarrhea to life-threatening inflammations of the colon,and include, but are not limited to, pseudomembranous colitis—severeinflammations of the colon, and colitis manifested by diarrhea, urgency,abdominal cramps, tenesmus, and fever among other symptoms. Diarrhea,when severe, causes dehydration and the medical complications associatedwith dehydration.

Further disorders associated with C. difficile, S. aureus and C.perfringens that can be treated with a compound of Formula II or acompound of Formula IIa or a salt thereof or a composition comprisingsuch a compound include, but are not limited to, bouts of waterydiarrhea, often with nausea and abdominal pain and cramping, colitis orpseudomembranous colitis, profuse watery diarrhea—10 or more bowelmovements a day, fever, often greater than 101° F., abdominal pain,which may be severe, blood or pus in the stool, nausea, dehydration, andweight loss

In certain embodiments, subjects can develop C. difficile, S. aureus andC. perfringens infections during or shortly after a course ofantibiotics, but signs and symptoms may not appear for weeks or evenmonths after treatment has stopped. The compounds and composition of theinvention are useful for long-term treatment and to prevent therecurrence of a bacterial infection or a disorder associated with abacterial infection.

Without being limited by theory, it is believed that when a subjecttakes an antibiotic to treat an infection, it often destroys thesebeneficial bacteria as well as the bacteria that are causing theillness. Without enough healthy bacteria, dangerous pathogens such as C.difficile can quickly grow out of control. Once C. difficile takes holdit can produce virulent toxins that attack the lining of the intestine.The toxins destroy cells and produce pseudomembranes—telltale patches(plaques) of inflammatory cells and decaying cellular debris on theinterior surface of the colon. Without being limited by theory, it isbelieved that almost any antibiotic can cause harmful bacteria toproliferate in the intestine, but ampicillin, amoxicillin, clindamycin,fluoroquinolones and cephalosporins are most often implicated in C.difficile infections. The use of broad-spectrum drugs that target a widerange of bacteria and the taking of antibiotics for a prolonged periodincrease the chance of infection. Other antimicrobials, includingantiviral and antifungal drugs and chemotherapy medications also canlead to an increased risk of infection with C. difficile.

Accordingly, the invention further encompasses compositions andformulations comprising a compound of formula II or a salt thereof thatare useful in treating infection, for example a bacterial infection,caused by abnormal growth of C. difficile, S. aureus and C. perfringensand/or treating or preventing disorders associated with abnormal growthof C. difficile, S. aureus and C. perfringens.

The invention also encompasses methods of treating infection, forexample a bacterial infection, including but not limited to, a bacterialinfection caused by C. difficile, S. aureus and C. perfringens and/ortreating or preventing disorders associated with C. difficile, S. aureusand C. perfringens comprising administering to a subject, preferably toa mammal in need of said treatment or prevention a therapeutically orprophylactically effective amount of a compound of Formula II or FormulaIIa or a salt thereof or a composition including such a compound.

In one embodiment, a composition of the invention comprising a compoundof Formula II or Formula IIa or a salt thereof and a pharmaceuticallyacceptable vehicle, is administered to a mammal, preferably a human,with a disorder associated with anaerobic and microaerophilic GI flora.

In another embodiment, a composition of the invention comprising acompound of Formula II or Formula IIa or a salt thereof and apharmaceutically acceptable vehicle, is administered to a mammal,preferably a human, with an infection caused by C. difficile, S. aureusand C. perfringens.

In another embodiment, a composition of the invention comprising acompound of Formula II or Formula IIa or a salt thereof and apharmaceutically acceptable vehicle, is administered to a mammal,preferably a human, with a C. difficile, S. aureus and/or C. perfringensassociated disorder.

In one embodiment, “treatment” or “treating” refers to an ameliorationof a disease or disorder, or at least one discernible symptom thereof,associated with a bacterial infection associated with abnormal growth ofanaerobic and microaerophilic GI flora or C. difficile, S. aureus and C.perfringens. In another embodiment, “treatment” or “treating” refers toan amelioration of at least one measurable physical parameter, notnecessarily discernible by the subject in need of the treatment. In yetanother embodiment, “treatment” or “treating” refers to inhibiting theprogression of a disease or disorder, either physically, for example,stabilization of a discernible symptom, physiologically, for example,stabilization of a physical parameter, or both. In yet anotherembodiment, “treatment” or “treating” refers to delaying the onset of adisease or disorder.

In certain embodiments, the compositions of the invention areadministered to a subject, preferably a human, as a preventative measureagainst such diseases. As used herein, “prevention” or “preventing”refers to a reduction of the risk of acquiring a given disease ordisorder. In a preferred mode of the embodiment, the compositions of theinvention are administered as a preventative measure to a subject,preferably a human having a predisposition to a bacterial infection ordisorder associated with abnormal growth of anaerobic andmicroaerophilic GI flora or C. difficile, S. aureus and/or C.perfringens.

Therapeutic/Prophylactic Administration and Compositions

Due to the activity of the compounds of Formula II and Formula IIa andsalts thereof, the compounds are advantageously useful in veterinary andhuman medicine. As described above, the compounds of the invention areuseful for the treatment or prevention of a disorder associated withanaerobic and microaerophilic GI flora, C. difficile, or a disorderassociated with C. difficile, S. aureus and/or C. perfringens.

In some embodiments, the subject has abnormal/altered gut flora but doesnot exhibit or manifest any physiological symptoms associated withabnormal growth of anaerobic and microaerophilic GI flora, C. difficile,S. aureus and/or C. perfringens, or a disorder associated with C.difficile, S. aureus and/or C. perfringens.

The invention encompasses methods of treatment and prophylaxis byadministration to a subject of a therapeutically or prophylacticallyeffective amount of a composition comprising a compound of Formula II.The invention also encompasses methods of treatment and prophylaxis bycontacting the gut or GI tract of a subject with a therapeutically orprophylactically effective amount of a compound of Formula II or FormulaIIa or a salt thereof.

The subject is a mammal, including, but not limited, to an animal such acow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat,rabbit, guinea pig, and is more preferably a human.

The compositions, which comprise a compound of Formula II, arepreferably administered orally. The compounds of the invention may alsobe administered by any other convenient route, for example, by infusionor bolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa) and may beadministered together with another biologically active agent.Administration can be systemic or local. Various delivery systems areknown, for example, encapsulation in liposomes, microparticles,microcapsules, capsules, etc., and can be used to administer a compoundof Formula II, Formula IIa, or a salt thereof. Methods of administrationinclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral,sublingual, intranasal, intracerebral, intravaginal, transdermal,rectally, by inhalation, or topically, particularly to the ears, nose,eyes, or skin. The preferred mode of administration is left to thediscretion of the practitioner, and will depend in-part upon the site ofthe medical condition.

In most instances, administration will not result in the release of thecompounds of the invention into the bloodstream. For example, inaccordance with one embodiment of the invention, after multiple doseoral administrations, low MCC levels of Formula II were detected inplasma, most of which fell below the limit of quantification. Bycontrast, fecal levels of Formula II in the studies were extremely high,exceeding 1,000 times its MIC values versus C. difficile.

However, in certain embodiments in may be useful to administer thecompounds of the invention systemically. Accordingly, in otherembodiments, administration will result in the release of the compoundsof the invention into the bloodstream.

In specific embodiments, it may be desirable to administer one or morecompounds of the invention locally to the area in need of treatment.This may be achieved, for example, and not by way of limitation, bylocal infusion during surgery, topical application, for example, inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of an atherosclerotic plaque tissue.

Pulmonary administration can also be employed, for example, by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Incertain embodiments, the compounds of the invention can be formulated asa suppository, with traditional binders and vehicles such astriglycerides.

In another embodiment, the compounds of the invention can be deliveredin a vesicle, in particular a liposome (see Langer, 1990, Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.).

In yet another embodiment, the compounds of the invention can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507 Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol.Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 71:105). In yet another embodiment, a controlled-releasesystem can be placed in proximity of the target of the compounds of theinvention, e.g., the liver, thus requiring only a fraction of thesystemic dose (see, e.g., Goodson, in Medical Applications of ControlledRelease, supra, vol. 2, pp. 115-138 (1984)). Other controlled-releasesystems discussed in the review by Langer, 1990, Science 249:1527-1533)may be used.

The compositions will contain a therapeutically or prophylacticallyeffective amount of a compound of Formula II, Formula IIa or a saltthereof, preferably in purified form, together with a suitable amount ofa pharmaceutically acceptable vehicle to provide the form for properadministration to the patient.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “vehicle” refers to a diluent, adjuvant, excipient, or carrier withwhich a compound of the invention is administered. Such pharmaceuticalvehicles can be liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. The pharmaceuticalvehicles can be saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea, and the like. In addition, auxiliary,stabilizing, thickening, lubricating and coloring agents may be used.When administered to a patient, the compounds of the invention andpharmaceutically acceptable vehicles are preferably sterile. Water is apreferred vehicle when the compound of the invention is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid vehicles, particularly forinjectable solutions. Suitable pharmaceutical vehicles also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The present compositions, if desired, canalso contain minor amounts of wetting or emulsifying agents, or pHbuffering agents.

The compounds of the invention can also be administered in the form ofsolutions, suspensions, emulsion, tablets, pills, pellets, capsules,capsules containing liquids, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, or any otherform suitable for use. In one embodiment, the pharmaceuticallyacceptable vehicle is a capsule. (See e.g., U.S. Pat. No. 5,698,155).Other examples of suitable pharmaceutical vehicles are described in“Remington's Pharmaceutical Sciences” by A.R. Gennaro.

In a certain embodiment, the compounds of Formula II are formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compounds of the invention for intravenous administration are solutionsin sterile isotonic aqueous buffer. Where necessary, the compositionsmay also include a solubilizing agent. Compositions for intravenousadministration may optionally include a local anesthetic such aslidocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compound ofthe invention is to be administered by infusion, it can be dispensed,for example, with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the compound of the invention isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

In other embodiments, the compositions of the invention can beadministered orally. Compositions for oral delivery may be in the formof tablets, lozenges, aqueous or oily suspensions, granules, powders,emulsions, capsules, syrups, or elixirs, for example. Orallyadministered compositions may contain one or more optionally agents, forexample, sweetening agents such as fructose, aspartame or saccharin;flavoring agents such as peppermint, oil of wintergreen, or cherry;coloring agents; and preserving agents, to provide a pharmaceuticallypalatable preparation. Moreover, where in tablet or pill form, thecompositions may be coated to delay disintegration and absorption in thegastrointestinal tract thereby providing a sustained action over anextended period of time. Selectively permeable membranes surrounding anosmotically active driving compound are also suitable for orallyadministered compounds of the invention. In these later platforms, fluidfrom the environment surrounding the capsule is imbibed by the drivingcompound, which swells to displace the agent or agent compositionthrough an aperture. These delivery platforms can provide an essentiallyzero-order delivery profile as opposed to the spiked profiles ofimmediate release formulations. A time delay material such as glycerolmonostearate or glycerol stearate may also be used. Oral compositionscan include standard vehicles such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Such vehicles are preferably of pharmaceutical grade.

The amount of a compound of the invention that will be effective in thetreatment of a particular disorder or condition disclosed herein willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro or in vivo assaysmay optionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the compositions will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances.

Suitable amounts of the compound of Formula II or IIa or a salt thereofin an oral composition for oral administration are generally about 0.001milligram to 4000 milligrams of a compound of the invention. Inpreferred embodiments of the invention, the amount is about 0.01milligram to 2000 milligrams, more preferably about 0.1 milligram to1000 milligrams, more preferably 0.5 milligram to 800 milligrams, andyet more preferably 1 milligram to 500 milligrams. In a preferredembodiment, the amount of compound of the invention or a salt thereof inthe composition is about 4000 milligrams of a compound. In a preferredembodiment, the amount of compound of the invention or a salt thereof inthe composition is about 2000 milligrams of a compound. In a preferredembodiment, the amount of compound of the invention or a salt thereof inthe composition is about 1000 milligrams of a compound. In a preferredembodiment, the amount of compound of the invention or a salt thereof inthe composition is about 500 milligrams of a compound. In anotherpreferred embodiment, the amount of compound of the invention or a saltthereof in the composition is about 250 milligrams of a compound. In apreferred embodiment, the amount of compound of the invention or a saltthereof in the composition is about 100 milligrams of a compound. In apreferred embodiment, the amount of compound of the invention or a saltthereof in the composition is about 50 milligrams of a compound. In apreferred embodiment, the amount of compound of the invention or a saltthereof in the composition is about 10 milligrams of a compound. Thedosage amounts described herein refer to total amounts administered;that is, if more than one compound of the invention is administered, thepreferred dosages correspond to the total amount of the compounds of theinvention administered. Oral compositions preferably contain 10% to 95%active ingredient by weight. In certain embodiments, the oral dosageregimen can include taking multiple oral dosages in a single day, forexample, taking the oral dose once a day, twice a day, three times aday, four times a day or more spaced out throughout the day.

In other embodiments, suitable dosage ranges for oral administration canbe generally about 0.001 milligram to 2000 milligrams of a compound ofthe invention per kilogram body weight. In specific preferredembodiments of the invention, the oral dose is 0.01 milligram to 1000milligrams per kilogram body weight, more preferably 0.1 milligram to500 milligrams per kilogram body weight, more preferably 0.5 milligramto 200 milligrams per kilogram body weight, and yet more preferably 1milligram to 100 milligrams per kilogram body weight. In a mostpreferred embodiment, the oral dose is 5 milligrams of a compound of theinvention per kilogram body weight. The dosage amounts described hereinrefer to total amounts administered; that is, if more than one compoundof the invention is administered, the preferred dosages correspond tothe total amount of the compounds of the invention administered. Oralcompositions preferably contain 10% to 95% active ingredient by weight.

Suitable dosage ranges for intravenous (i.v.) administration are 0.01milligram to 100 milligrams per kilogram body weight, 0.1 milligram to35 milligrams per kilogram body weight, and 1 milligram to 10 milligramsper kilogram body weight. Suitable dosage ranges for intranasaladministration are generally about 0.01 pg/kg body weight to 1 mg/kgbody weight. Suppositories generally contain 0.01 milligram to 50milligrams of a compound of the invention per kilogram body weight andcomprise active ingredient in the range of 0.5% to 10% by weight.Recommended dosages for intradermal, intramuscular, intraperitoneal,subcutaneous, epidural, sublingual, intracerebral, intravaginal,transdermal administration or administration by inhalation are in therange of 0.001 milligram to 200 milligrams per kilogram of body weight.The compounds of the invention can also be incorporated into a topicalformulation, for example, an ointment, suave, cream, or oil. Suitabledoses of the compounds of the invention for topical administration arein the range of 0.001 milligram to 2000 milligram, depending on the areato which the compound is administered. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. Such animal models and systems are well known in theart.

The invention also provides pharmaceutical packs or kits comprising oneor more containers filled with one or more compounds of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In a certain embodiment, the kit contains more than onecompound of the invention. Other methods will be known to the skilledartisan and are within the scope of the invention.

EXAMPLES

The following examples are provided by way of describing specificembodiments of the present invention without intending to limit thescope of the invention in any way.

Example 1 Formula II Preparation

At 0° C., LiOH (42 mg) in 12 mL of MeOH was added to 783 mg of OPT-80.The Solution was kept at 0° C. for 5 h, and then quenched by addition ofsaturated NH₄Cl solution. The resulting mixture was extracted withchloroform. The organic layer was separated, dried and concentrated. Thecrude was purified by silica gel column chromatography with CH₂Cl₂:CH₃OH10:1 as eluents. The desired product (154 mg) was isolated with 97.05%purity by HPLC.

Analytical data of Formula II.

MS m/z (ESI) 1004.4 (M+NH₄)⁺;

¹H NMR (400 MHz, CDCl₃) δ 7.12 (d, J=11.4 Hz, 1H), 6.59 (dd, J=14.7,11.4 Hz, 1H), 5.85 (br s, 1H), 5.83 (ddd, J=14.7, 10.6, 4.0 Hz, 1H),5.47 (dd, J=8.1, 7.6 Hz, 1H), 5.15 (dd, J=9.6, 9.6 Hz, 1H), 4.98 (d,J=10.6 Hz, 1H), 4.76−4.70 (m, 1H), 4.69 (d, J=11.6 Hz, 1H), 4.63 (br s,1H), 4.62 (br s, 1H), 4.39 (d, J=11.6 Hz, 1H), 4.29−4.23 (m, 1H),4.07−3.98 (m, 2H), 3.72−3.48 (m, 5H), 3.62 (s, 3H), 3.17 (d, J=5.8 Hz,1H), 3.14−2.96 (m, 2H), 2.81−2.45 (m, 5H), 2.33−2.25 (m, 1H), 1.92 (s,3H), 1.81 (s, 3H), 1.67 (s, 3H), 1.64−1.57 (m, 1H), 1.34 (d, J=6.1 Hz,3H), 1.27 (s, 3H), 1.21 (t, J=7.3 Hz, 3H), 1.19 (d, J=6.8 Hz, 3H), 1.05(s, 3H), 0.83 (t, J=7.3 Hz, 3H);

¹³C NMR (100 MHz, CDCl₃) δ 170.1, 168.9, 157.6, 152.7, 144.2, 140.9,140.5, 136.9, 136.3, 134.5, 134.4, 129.1, 127.9, 125.3, 123.3, 113.9,107.6, 107.2, 101.9, 94.6, 92.4, 82.3, 78.2, 76.9, 74.6, 74.5, 72.7,71.9, 71.8, 71.4, 70.1, 69.2, 63.7, 62.3, 41.9, 36.9, 28.9, 28.4, 26.2,25.9, 18.5, 17.8, 17.4, 17.2, 15.5, 14.1, 13.9, 11.2. Aqueoussolubility: ˜300 mg/mL

HPLC Chromatography analysis was performed using Walters 2695 separationmodule, with Walters 2487 detector.

Column: Agilent, ZORBAX Eclipse XDB-C8 4.6×150 mm 3.5 micron

Detector wavelength: 230 nm

Mobile phase A is water, 0.1% TFA, mobile phase B is acetonitrile 0.05%TFA

Gradient:

Time(min) Flow(ml/min) A B 1 60 40 3 1 50 50 14 1 39 61 14.5 1 60 40 191 60 40Formula II came out at 4.81 min.

The 4.81 min peak was also found in the HPLC diagram of the crudematerial of OPT-80 production after fermentation.

Example 2 Formula II Biological Activity

Bacterial Strains:

Most bacterial strains were purchased from American Type CultureCollection (Manassas, Va.). A few clinical isolates of Staphylococcusand vancomycin resistant Enterococci were purchased from Christiana CareHealth System (Wilmington, Del.) and were identified by Biomerieux APIidentification system (Durham, N.C.).

Antimicrobial Compounds:

OPT-80 and Formula II were produced by Optimer Pharmaceuticals.Vancomycin was purchased from Sigma-Aldrich (St. Louis, Mo.).

Formula II Profiling

The MIC of OPT-80 and its major metabolite, Formula II, againstanaerobic and aerobic GI bacteria was determined using the CLSI standardsusceptibility testing methods.

Agar Dilution Method: (1)

This method was used for anaerobic organisms (Bacteroides, Clostridium,Bifidiobacteria, Prevotella, Fusobacterium, Peptococcus,Peptostreptococcus, Veillonella) and microaerophilic Lactobacillus spp,all of which were purchased form American Type Culture Collection(ATCC):

1. Two fold dilutions of compounds were made in Brucella agarsupplemented with hemin, vitamin K and 5% laked sheep blood to achieve afinal concentration range from 0.016 μg/mL to 16 μg/mL.

2. Using the direct colony suspension method, bacterial inocula wereprepared with densities close to a 0.5 McFarland standard (˜10⁸ CFU/mL),followed by spotting of the inocula onto agar plates using a Steersreplicator to deliver ˜10⁵ CFU/spot.

3. Plates were incubated in an anaerobic chamber at 35° C. for 48 hours.For the Lactobacillus strains, Mueller Hinton Agar and an incubationatmosphere of 5% CO₂ were used.

4. The lowest concentration of an antibiotic showing no growth or markedreduction in growth was read as the MIC.

Microbroth Dilution Method:

This susceptibility method was used for aerobic and facultativeorganisms (Escherichia, Pseudomonas, Salmonella, Haemophilus.Streptococcus, Staphylococcus and Enterococcus):

1. Two fold serial dilutions of compounds were made in cation-adjustedMueller Hinton broth to achieve a concentration range from 0.125 μg/mLto 64 μg/mL.

2. Using the direct colony suspension method, bacterial inocula at 0.5McFarland standard were diluted and inoculated into each microtiterplate to a final density of approximately 5×10⁵ CFU/mL (or 5×10⁴CFU/well).

3. The microtiter plates were incubated for 16-24 hours (depending onthe organism) at 35° C. in ambient air.

4. The MIC was read as the lowest concentration at which there was novisible growth.

Effect of Fecal Material on the In Vitro Activity of OPT-80 and FormulaII:

The in vitro activity of OPT-80, Formula II, and vancomycin against C.difficile ATCC 700057 was measured in the presence and absence of fecesusing microbroth dilution susceptibility testing methods according tothe CLSI guidelines as is recommended for the B. fragilis. The Brucellabroth media (BB) used for the experiment was supplemented with hemin (5μg/mL) and vitamin K (1). The fecal slurry was prepared according to themethod described by Swanson et al (Antimicrobial Agents andChemotherapy. 35(6): 1108-1111; J of Antibiotics. XLII (1): 94-101).

1. Briefly, a 5% fecal slurry was prepared by adding 5.5 gram of freshhuman fecal sample into 110 ml BB, followed by homogenization forapproximately 10 minutes.

2. The slurry was aliquoted into separate tubes containing 2× thedesired top concentrations of the following drugs: OPT-80, Formula II,vancomycin (VAN), or no drug (Preparations A-F and I, respectively).

3. Similar preparations of the above drugs in BB without feces were alsoprepared as controls for the experimental procedure (Preparations E-Gand J, respectively).

Preparation A: Feces-supplemented BB+32 μg/mL OPT-80

Preparation B: Feces-supplemented BB+256 μg/mL Formula II

Preparation C: Feces-supplemented BB+256 μg/mL VAN

Preparation E: Unsupplemented BB+32 μg/mL OPT-80

Preparation F: Unsupplemented BB+256 μg/mL Formula II

Preparation G: Unsupplemented BB+256 μg/mL VAN

Preparation I: Feces-supplemented BB, drug-free

Preparation J: Unsupplemented BB, drug-free

Following 30 minutes incubation at room temperature, all preparationswere centrifuged to remove solid material, filtered once through a 1.2μm microfiber glass pre-filter, and filtered three times through 0.2 μmfilter devices.

The filtrate materials from preparations A-C were serially dilutedacross the length of 96-well microtiter plates using the 5% fecal slurryfiltrate (i.e. Preparation I) as the diluent.

The filtrate materials from Preparation E-G were serially diluted acrossthe length of 96 well microtiter plates using BB (Preparation J) as thediluent.

The plates were transferred to an anaerobic glove box and were allowedto equilibrate for 4 hours to the anaerobic environment.

Bacterial inocula were prepared by harvesting bacteria from overnightculture and preparing a 0.5 McFarland suspension into feces-supplementedor unsupplemented BB.

Prepared inocula were then added to plates containing serially diluteddrugs, resulting in a 1:2 dilution of drugs to give the desired finalconcentration.

The microtiter plates were incubated for ˜48 hours at 35° C. underanaerobic conditions, and the MIC was read as the lowest drugconcentration at which there was no visible growth.

Results of Formula II Profiling

Formula II, similar to the parent compound, demonstrated antimicrobialactivity against some gram positive bacteria (including C. difficile, C.perfringenes, B. longum, F. magna, P. asaccharolyticus, P. anaerobius,M. micros and the microaerophilic L. casei), albeit with MIC values thatwere four to sixteen fold greater than those of OPT-80. With regards toother gram positive organisms such as S. aureus, E. faecium and L.rhamnosus, Formula II demonstrated in vitro activity with MICs of >16μg/mL. This clearly demonstrates Formula II can be used as a very narrowspectrum antibiotic against C. difficile.

TABLE 1 Raw MIC data (μg/mL) for OPT-80 and Formula II against grampositive bacterial strains representative of GI flora. Formula GramPositive Organisms II OPT-80 Bifidiobacterium longum, ATCC 15707 1 0.125Clostridium difficile, ATCC 700057 2 0.25 Clostridium difficile, ATCC43255 4 0.5 Clostridium perfringens, ATCC 13124 1 0.125 Enterococcusfaecium, ATCC 19434 >64 4 Enterococcus faecium, Vancomycin >64 8resistant clinical strain ORG#360 Enterococcus faecium, Vancomycin 64 2resistant clinical strain ORG#364 Enterococcus faecium, Vancomycin >64 4resistant clinical strain ORG#534 Finegoldia (formerly Peptococcus) 8 1magna, ATCC 29328 Lactobacillus. casei, ATCC 393 8 2 Lactobacillusacidophilus, ATCC 4356 >16 >16 Lactobacillus rhamnosus, ATCC 7496 >16 8Micromonas (formerly Peptostreptococcus) 1 0.125 micros, ATCC 33270Peptoniphilus (formerly Peptococcus) 4 1 asaccharolyticus, ATCC 29743Peptostreptococcus anaerobius, ATCC 27337 0.25 0.016 Staphylococcusaureus, ATCC 29213 >64 8 Staphylococcus aureus, ATCC 33591 >64 4(Methicilin resistant) Staphylococcus aureus, Methicilin >64 8 resistantclinical strain ORG#578 Staphylococcus epidennidis, clinical 64 4 strainORG#365 Streptococcus pneumoniae, ATCC 49619 >64 64 Streptococcuspyogenes, ATCC 19615 >64 16

In the phase 2A study of OPT-80 in CDAD patients who were administeredOPT-80 at 200 mg bid (the intended dosing regimen), fecal levels of theparent OPT-80 were 1433±975 μg/g, while fecal levels of the metaboliteFormula II were 760±373 μg/g (6). Thus, after oral administration, bothparent and metabolite are present at levels far in excess of their MICvalues versus C. difficile (FIG. 1).

Formula II is present in the gut after oral dosing with OPT-80 at levelsfar in excess (1,000 times) of their MIC values vs. C. difficile.

TABLE 2 MIC data for OPT-80 and Formula II for a panel of aerobicbacteria Aerobic Panel MIC (μg/mL) ORG 84 ORG 97 ORG 98 ORG 83 E.faecium ORG 85 S. aureus S. aureus ORG 101 E. faecalis ATCC E. faecium(MRSA) (MSSA) ORG 99 S. pyogenes ATCC 700221 ATCC ATCC ATCC S.epidermidis ATCC Compound 29212 VRE 19434 33591 29213 ATCC 12228 19615OPT-80 2-4 4 8 8 8 4 16-32 Formula 16-64 >32 >64 >64 >64 >32 >32 II

TABLE 3 MIC data for OPT-80 and Formula II for a panel of anaerobicbacteria Aerobic Panel MIC (μg/mL) ORG 830 C. difficile ORG 150 B.fragilis Compound ATCC 700057 25285 OPT-80 0.030, 0.06 >16 Formula II0.5-1 >16

Effect of Fecal Material on In Vitro Activity of OPT-80 and Formula II:

The effect of fecal material on the antimicrobial activity of OPT-80,Formula II and vancomycin, which is the only FDA approved treatment forCDAD, was evaluated vs. C. difficile ATCC strain 700057 (Table 3).

In the presence of 5% fecal material, a similar fold increase in MICvalues was observed for all three drugs; OPT-80 (2 μg/ml; eight fold),Formula II (4 μg/ml; four fold) and vancomycin (8 μg/ml; four fold). Thein vitro data demonstrates that even in the presence of fecal materialboth the metabolite and OPT-80 have better or similar in vitro activitycompared to vancomycin, the approved treatment for CDAD. Furthermore,the average fecal concentration of OPT-80 following oral administrationhas been shown to be over 1,400 μg/g of stool (6), a level far in excessof the MIC even in the presence of fecal material.

TABLE 4 In vitro activity of drugs in presence or absence of fecalmaterial vs. C. difficile ATCC strain 700057 MIC (μg/mL) Diluent OPT-80FORMULA II Vancomycin Brucella broth only 0.25 1 2 Brucella broth withfeces 2 4 8 Fold increase in MIC 8x 4x 4x

All references discussed above are herein incorporated by reference intheir entirety for all purposes. While this invention has beenparticularly shown and described with references to preferredembodiments thereof, it will be understood by those skilled in the artthat various changes in form and details may be made therein withoutdeparting from the spirit and scope of the invention as defined by theappended claims.

What is claimed is:
 1. A method of treating a Clostridium difficileinfection in a subject in need thereof comprising administering to thesubject a composition comprising a therapeutically effective amount of acompound of Formula II:

in free form or in the form of a pharmaceutically acceptable saltthereof.
 2. The method of claim 1, wherein the infection is agastrointestinal infection.
 3. The method of claim 2, wherein thesubject is human.
 4. The method of claim 3, wherein the subject haspreviously been treated for a Clostridium difficile infection with acompound other than Formula II.
 5. The method of claim 3, wherein thesubject is suffering from Clostridium difficile associated diarrhea. 6.The method of claim 3, wherein the subject is suffering frompseudo-membranous colitis.
 7. The method of claim 6, wherein the subjectis suffering from antibiotic associated diarrhea.
 8. The method of claim2, wherein the route of administration is oral.
 9. The method of claim1, wherein the dosage is 0.1 milligrams to 200 milligrams per kilogramof the subject's body weight.
 10. The method of claim 1, wherein thedosage is 1 milligram to 200 milligrams per kilogram of the subject'sbody weight.
 11. The method of claim 1, wherein the dosage is 1milligram to 100 milligrams per kilogram of the subject's body weight.12. The method of claim 1, wherein the dosage is 2 milligrams to 50milligrams per kilogram of the subject's body weight.